Fusion antigen used as vaccine

ABSTRACT

The present invention mainly provides a fusion antigen specific for a target cell comprising a ligand moiety which is capable of reacting, recognizing or binding to the receptors on the target cell, a  Pseudomonas  exotoxin A translocation domain II, an antigenic moiety, and a carboxyl terminal moiety which permits combination of the fusion antigen to the endoplasmic reticulum (ER) membrane of the target cell. A method of immunizing an animal using the fusion antigen is also provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention mainly relates to a fusion antigen. Moreparticularly, the invention mainly relates to a fusion antigen used as aT-cell vaccine.

[0003] 2. Description of the Related Art

[0004] The mechanism of immunization in an animal comprises humoralimmunity and cell-mediated immune response.

[0005] Humoral immunity mainly relates to the production of antibodies.Antibodies can provide protection from pathogens or their toxic productsby binding to them and thereby blocking their access to cells that maybe infected or destroyed. Antibodies can also trigger a phagocytic cellto ingest and destroy the pathogen, such as a bacterium. The thirdfunction of antibodies is to activate a system of plasma proteins, knownas a complement, that can directly destroy bacteria.

[0006] Cell-mediated immune reactions depend on direct interactionsbetween T-lymphocytes and cells bearing the antigen that T-cellsrecognize. These cells recognize body cells infected with viruses, whichreplicate inside cells using the synthetic machinery of the cell itself.Antigens derived in the replication of a virus, however,are present onthe surface of infected cells (by MHC class I), where they arerecognized by cytotoxic T-cells (CD8⁺ T-cells) and these may thencontrol the infection by killing the cells before the replication of avirus is complete.

[0007] Vaccines for prophylaxis of viral infections are usually liveattenuated organisms with reduced pathogenicity that would stimulateprotective immunity. Foreign proteins of a live virus that is used as alive attenuated vaccine, are recognized and processed in the endoplasmicreticulum (ER) lumen of antigen presenting cells (APCs) when the virusreplicates to form an endogenous processing peptide. The processincludes antigen modification and proper digestion. However, a liveattenuated vaccine has a quite strong tendency to recover toxicity. Forexample, the toxicity of infectious laryngotracheitis virus (ILTV)recovers both in vaccine or attenuated strains. Besides, multiplepassages of a virus should be operated; therefore, the ability to evokean immune response is discredited. It is a time-consuming job to developa live attenuated vaccine.

[0008] To prevent the recovery of a live attenuated vaccine, genedeficient vaccines are developed, such as Aujeszky's disease vaccines,gI negative vaccines, and PRV marker vaccines.

[0009] In another aspect, recombinant subunit vaccines and DNA vaccinesare also disclosed. Viruses or bacteria of vaccina or fowlpox are usedas vectors for carrying the genes of the antigens. Through recombinantDNA technology, the time for development of a good vaccine is reducedand multiple serotypes of vaccine can be achieved at the same time.Examples of such vaccines are fowlpoxvirus and Salmonella vector systemsand Syntro Vet (US) gene recombinant vaccines. On the other hand, when amicroorganism, especially an RNA virus, is used as a vector, themicroorganism would derive a new species or a new strain. The safety ofsuch vaccines is challenged. Besides, recombinant subunit vaccines areusually helpless in triggering a cell-mediated immune response. They areexogenous antigens, which are taken into macrophages, dendritic cellsand B lymphocytes. Peptides from exogenous antigens are generated afterthe internalization of the antigens within APCs via fluid phasepinocytosis or membrane-bound receptors. The peptides are generated inthe endosomal compartments of the APCs and sorted by empty MHC class IImolecules to form peptide-MHC class II complexes based on the affinitiesbetween the MHC class II molecules and the peptides. The peptide-MHCclass II complexes are then translocated to the surface of the APCswhere they are recognized by CD4⁺ T-cells. However, small subunitproteins recognized by CD8⁺ T-cells cannot be used efficiently asvaccines because, once parenterally administered, they are internalizedin endosomal compartments where they are likely to be either extensivelydegraded or fail to interact with the MHC class I pathway. Furthermore,CD4⁺ cells (Th cells) can both activate humoral immunity andcell-mediated immune response by Th1 and Th2 helper T-cells,respectively. Th1 and Th2 cells regulate each other for the balance ofhumoral immunity and cell-mediated immune response. Therefore, if onlyhumoral antibodies will produce in all the immune responses, viralinfection will be less controlled because of over sensitization of animmune system. Fortunately, it is now possible to envisage thepreparation of safe T-cell vaccines able to induce protectivecell-mediated immunity against all viruses (Constantin A. Bona, et al.1998. Immunology today vol 19. 126-131).

[0010] Vaccines for virus-infecting immunological cells such as T-cell,B-cell, dendritic cell, monocyte, and macrophage still remain to bedeveloped. Examples of such viruses are porcine reproductive andrespiratory syndrome virus, Circovirus type II, and humanimmunodeficiency virus. Such viruses shut down the ability ofrecognition of foreign proteins as antigens in the antigen presentingcells. The immunological cells cannot function and carry the viruses.This kind of infection usually leads to acute damage to the animalinfected. The animals that have been infected are easily infected byother pathogens. A recent report shows that cytotoxic T-cells (CTLs) areessential for controlling HIV infection. (Hanne G-S et al 2000. Journalof virology vol 74, No. 4. p. 1694-1703). It is a pity that a usefulvaccine for virus-infecting immunological cells is still lacking.

[0011] In particular, porcine reproductive and respiratory syndromevirus (PRRSV) results in high losses in animal husbandry every year. Thevirus infects macrophages (in the alveolar and spleen), brain microgliaand monocytes, and exists in the blood and organs of the infectedanimals. Antibodies have little effect on the virus and even stimulatesmutations of the virus. In the mechanism of antibody dependentenhancement (ADE), the use of antibodies lead to more severe infections.About 50 to 80% of pigs are infected by such virus. Generally, theanimals infected by the virus have no significant symptoms, but theimmunity of the infected animals will be reduced. This leads to adecrease of weight gain and an increase in death rate. PRRSV is an RNAvirus. Not only animals, but also ducks would be infected by PRRSV. Alive attenuated vaccine against PRRSV was developed. However, mutationof the viruses in the live vaccine quite often occurs. To develop a safevaccine is desired.

SUMMARY OF THE INVENTION

[0012] The present invention provides a fusion antigen which can be usedas a T-cell vaccine, and preferably, as a T-cell vaccine against thevirus-infecting immunological cells. The invention is characterized bythe design of carboxyl terminal moiety of the fusion antigen.

[0013] One objective of the invention is to provide a fusion antigenspecific for a target cell comprising an antigenic moiety, a ligandmoiety which is capable of reacting, recognizing or binding to thereceptors on the target cell, a Pseudomonas exotoxin A translocationdomain II, and a carboxyl terminal moiety which permits retention of thefusion antigen in the endoplasmic reticulum (ER) membrane of the targetcell.

[0014] Another objective of the invention is to provide a pharmaceuticalcomposition comprising the fusion antigen of the invention together witha pharmaceutically acceptable carrier.

[0015] Still another objective of the invention is to provide a methodof immunizing an animal comprising the steps of:

[0016] (a) providing a fusion antigen specific for a target cellcomprising an antigenic moiety, a ligand moiety which is capable ofreacting, recognizing or binding to a receptor on the target cell, aPseudomonas exotoxin A translocation domain II, and a carboxyl terminalmoiety which permits retention of the fusion antigen in the endoplasmicreticulum (ER) membrane of the target cell; and

[0017] (b) inoculating the animal with the fusion antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates the construction of PRRSV ORF 7 fusion antigenaccording to Example 1.

[0019]FIG. 2 illustrates the gene sequence of PRRSV ORF 7.

[0020]FIG. 3 illustrates the construction of PE-PRRS7-D in pET23aprotein expression plasmid.

[0021]FIG. 4 illustrates the construction of pET23-3KDEL.

[0022]FIG. 5 illustrates the construction of PRRSV ORF 7 fusion antigen.

[0023]FIG. 6 illustrates the SDS-PAGE detection of protein purificationand quantitative analysis for the sample of PE-DGD protein. The PE-DGDprotein gene of a PE(ΔIII) and PRRSV ORF-7 chimeria in pET plasmidsystem is expressed in BL21(DE3)plys by IPTG induction. The SDS-PAGEmaps of the total bacterial proteins are shown after the IPTG inductionsample (Lane A) and the urea extraction sample (Lane B or Lane 4). Onestrong staining band located at 80 K Da is PE-DGD protein. Lanes 1, 2and 3 are the samples of standard BSA protein loaded with the amounts of1000 ng, 300 ng and 100 ng, respectively. Lanes 4, 5 and 6 are thesamples of PE-DCD urea extraction protein with the amounts of 1 μL, 0.1μL and 0.01 μL, respectively.

[0024]FIG. 7 illustrates the SDS-PAGE detection of protein purificationand quantitative analysis for the sample of PE-DGDK protein. The PE-DCDKprotein gene of a PE(ΔIII) and PRRSV ORF-7 chimeria in pET plasmidsystem is expressed in BL21(DE3)plys by IPTG induction. The SDS-PAGEmaps of the total bacterial proteins are shown for the samples before(Lane A) and after IPTG induction (Lane B) and the urea extractionsample (Lane C or Lane 4). One strong staining band located at 80 K Dais PE-DGDK protein. Lanes 1, 2 and 3 are the samples of standard BSAprotein loaded with the amounts of 1000 ng, 300 ng and 100 ng,respectively. Lanes 4, 5 and 6 are the samples of PE-DGDK ureaextraction protein with the amounts of 1 μL, 0.1 μL and 0.01 μL,respectively.

[0025]FIG. 8 illustrates the detection limit of RT-PCR with BM-ORF7primers in pig blood leukocyte sample. The total RNA of 100 μL pig bloodleukocyte samples with 1 μL 3-fold serial dilution of PRRS virus (10⁶TCID₅₀/ml) were respectively extracted. The PRRSV detection limit wasdetermined by RT-PCR running with BM-ORF7 primers and 10/25 volume RNAtemplate. The RT-PCR products of the spike samples containingapproximately 300, 100, 30, 10, 3, 1 (TCID₅₀/ml) of PRRS, were loaded asLane 1, 2, 3, 4, 5, 6, respectively, and running at 2% agarose gel inTBE buffer.

[0026]FIG. 9 illustrates the result of the real time PCR analysis ofPRRSV by BM-primers.

[0027]FIG. 10 illustrates the result of the comparison of the detectionlimit of real-time PCR with tradition RT-PCR.

[0028]FIG. 11 illustrates the result of the detection of PRRSV by RT-PCRof five female pigs, wherein M represents the DNA marker and Prepresents the positive control.

[0029]FIG. 12 illustrates the result of the detection of PRRSV by RT-PCRbefore a challenge of 15 piglets, wherein M represents the DNA markerand P represents the positive control.

[0030]FIG. 13 illustrates the result of the detection of PRRSV by RT-PCR3 days after the challenge of 15 piglets, wherein M represents the DNAmarker and P represents the positive control.

[0031]FIG. 14 illustrates the result of the detection of PRRSV by RT-PCR7 days after the challenge of 15 piglets, wherein M represents the DNAmarker and P represents the positive control.

[0032]FIG. 15 illustrates the result of the detection of PRRSV by RT-PCR14 days after the challenge of 15 piglets, wherein M represents the DNAmarker and P represents the positive control.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The invention provides a fusion antigen specific for a targetcell comprising an antigenic moiety, a ligand moiety which is capable ofreacting, recognizing or binding to a receptor on the target cell, aPseudomonas exotoxin A translocation domain II, and a carboxyl terminalmoiety which permits retention of the fusion antigen in the endoplasmicreticulum (ER) membrane of the target cell.

[0034] As used herein, the term “fusion antigen” refers to a recombinantprotein which can evoke an immune response in an animal. Preferably, thefusion antigen comprises epitopes for evoking an immune responsedirectly and other portions for enhancing an immune response such asmediating delivery, transporting, processing, and expressing or forequipment of multiple functions.

[0035] Preferably, the target cell is an antigen presenting cell. Morepreferably, the target cell is selected from the group consisting ofT-cells, B-cells, dendritic cells, monocytes, and macrophages.

[0036] As used herein, the term “an antigenic moiety” refers to apeptide fragment that can evoke an immune response. In one embodiment ofthe invention, the antigenic moiety is an epitope. According to theinvention, the antigenic moiety is a protein of a pathogenic species,which can highly activate an immune response. Such proteins comprise,for example, but are not limited to, coat proteins, nucleoproteins orcell membrane proteins. The antigenic moiety can be a peptide cloneddirectly from the pathogenic species as well as a recombinant proteinmodified by artisans skilled in the field for enhancing the ability toevoke an immune response, for being manufactured more conveniently andfor being delivered more easily. Preferably, the antigenic moiety isderived from porcine reproductive and respiratory syndrome virus(PRRSV), Circovirus type II, or human immunodeficiency virus.Preferably, the antigenic moiety may be PRRSV ORF 1, 2, 3, 4, 5, 6, or7. In one more preferred embodiment of the invention, the antigenicmoiety is PRRSV ORF 7. For evoking a more severe immune response, theantigenic moiety comprises at least one antigenic unit and the adjacentantigenic unit is connected by a bridge region. According to theinvention, the bridge region may be a small fragment of peptide thatevokes little immune response to prevent the immune system fromrecognizing it.

[0037] As used herein, the term “ligand moiety” refers generally to allmolecules which are capable of reacting, recognizing or binding to thereceptor on a target cell. Examples of such receptors include, but arenot limited to, antibody receptors, growth factor receptors, lymphokinereceptors, cytokine receptors, hormone receptors and the like. In someembodiments of the invention, the receptor for binding to the ligandmoiety is selected from the group consisting of TGFα receptors, IL2receptors, IL4 receptors, IL6 receptors, IGF 1 receptors, CD4 receptors,IL18 receptors, IL 12 receptors, EGF receptors, LDL receptors andα2-macroglobulin receptors. The ligand moiety has an ability of bindingto the cell membrane of the target cell for anchoring the fusion antigento the target cell. The immune system is initiated by the fusionantigen's binding to the receptors on the target cell. Preferably, theligand moiety is a Pseudomonas exotoxin A binding domain I. Pseudomonasexotoxin A (PE) is a single polypeptide chain of 613 amino acids. X-raycrystallographic studies and mutational analysis of the PE molecule showthat PE consists of three domains: an amino terminal cell receptorbinding domain (Domain I); a middle translocation domain (Domain II);and a carboxyl terminal activity domain (Domain III) (see U.S. Pat. No.:5,705,163, which is incorporated into references).

[0038] As used herein, the term “Pseudomonas exotoxin A binding domainI” refers to a peptide fragment that has the same sequence as the aminoterminal cell receptor binding domain of Pseudomonas exotoxin A or afunctionally equivalent fragment. The amino terminal cell receptorbinding domain of Pseudomonas exotoxin A comprises two sub-domains,designated as domain Ia and domain Ib. The configuration of domain Iaand domain Ib can bind to a LDL receptor or α2-macroglobulin receptor ona cell surface. As used herein, the term “Pseudomonas exotoxin A bindingdomain II” refers to a peptide fragment that has the same sequence asthe middle translocation domain of Pseudomonas exotoxin A or afunctionally equivalent fragment. The Pseudomonas exotoxin Atranslocation domain II has an ability to translocate the fusion antigeninto the cytoplasm of the target cell. The fusion antigen istranslocated into the target cell after attaching to the target cellmembrane.

[0039] As used herein, the term “carboxyl terminal moiety which permitsretention of the fusion antigen to the endoplasmic reticulum (ER)membrane of a target cell” refers to a peptide fragment that enables thefusion antigen to bind to the ER membrane and to retain it in the ERlumen. In one embodiment of the invention, the carboxyl terminal moietycomprises, in a direction from the amino terminus to the carboxylterminus, the following amino acid residues:

R¹-R²-R³-R⁴-(R⁵)_(n)

[0040] Wherein,

[0041] R¹ is a positively charged amino acid residue;

[0042] R² is a negatively charged amino acid residue;

[0043] R³ is a negatively charged amino acid residue;

[0044] R⁴ is L;

[0045] R⁵ is a positively charged amino acid residue; and

[0046] n is 0 or 1.

[0047] Preferably, the carboxyl terminal moiety is a member of the KDELfamily protein. As used herein, the term “KDEL family protein” refers toa group of proteins, which has a similar carboxyl end binding to the ERmembrane of a cell and further has an ability for retention of suchprotein in the ER lumen. Generally, the length of the carboxyl endranges from 4 to 16 residues. As discussed in U.S. Pat. No. 5,705,163(which is incorporated into the references), the amino residues at thecarboxyl end of a KDEL family protein, particularly those in the lastfive amino acids, are important. As shown in the studies on the similarsequences present in different molecules and performing a specificbiological function, a sequence that retains a newly formed proteinwithin the endoplasmic reticulum is LysAspGluLeu. These findings suggestthat the sequence at the carboxyl end of the fusion antigen according tothe invention acts as some type of recognition sequence to assisttranslocation of the fusion antigen from an endocytic compartment intothe ER and retains it in the lumen. In a preferred embodiment, thecarboxyl terminal moiety comprises a sequence of KDEL. In a morepreferred embodiment, the carboxyl terminal moiety comprises a sequenceof KKDL-RDEL-KDEL.

[0048] The invention is characterized by the design of carboxyl terminalmoiety, which enables the fusion antigen to be processed in the ER ofthe target cell for combining with MHC class I molecules and beingrecognized by T-cells. The fusion antigen according to the invention isuseful in triggering cell-mediated immune reactions.

[0049] According to the invention, the fusion antigen is used for theimmunization of animals. One objective of the invention is to provide apharmaceutical composition comprising the fusion antigen of theinvention together with a pharmaceutical acceptable carrier. Preferably,the pharmaceutical composition is a T-cell vaccine.

[0050] As used herein, the term “T-cell vaccine” refers to a vaccinethat can protect a subject from infection by activating cell-mediatedimmune response. The crucial role of the T-cell vaccine is cytotoxicT-cell (also known as cytotoxic T lymphocyte, CD8⁺T-cell, and CTL) andmemory T-cells (T_(cm) and T_(em)).

[0051] The present invention also provides a method of immunizing ananimal comprising the steps of:

[0052] (a) providing a fusion antigen specific for a target cellcomprising an antigenic moiety, a ligand moiety which is capable ofreacting, recognizing or binding to a receptor on the target cell, aPseudomonas exotoxin A translocation domain II, and a carboxyl terminalmoiety which permits retention of the fusion antigen in the endoplasmicreticulum (ER) membrane of the target cell; and

[0053] (b) inoculating the animal with the fusion antigen.

[0054] In the Step (b) of the method, the animals may be inoculated withthe fusion antigen in any way known to artisans skilled in this field.For example, the fusion antigen may be delivered by injection or in aform of oral vaccine. Booster shots are optional, if necessary.Preferably, the inoculation is performed before infection. Newly bornanimals, even an embryo, may also be inoculated with the fusion antigento produce better immunity.

[0055] According to the invention, the following actions occur duringthe process of the response to the immunization:

[0056] (c) the target cell membrane binds to the ligand moiety foranchoring the fusion antigen to the target cell;

[0057] (d) the fusion antigen is translocated into the cytoplasm of thetarget cell by the Pseudomonas exotoxin A translocation domain II;

[0058] (e) the ER membrane of the target cell binds to the carboxylterminal moiety of the fusion antigen for retention of the fusionantigen in the ER lumen;

[0059] (f) the antigenic moiety is processed in the ER lumen;

[0060] (g) the processed antigenic moiety binds with a MHC class Imolecule;

[0061] (h) the processed antigenic moiety is carried by the MHC Class Imolecule to the target cell surface;

[0062] (i) the processed antigenic moiety carried by the MHC class Imolecule by CD8⁺ T-cell is recognized to obtain an immune message; and

[0063] (j) the immune message is stored by memory T-cells for immunizingthe animal.

[0064] In Action (c), the ligand moiety of the fusion antigen leads thefusion antigen to bind to the receptors on the target cell membrane foranchoring the fusion antigen to the target cell.

[0065] In Action (d), the fusion antigen is translocated into thecytoplasm of the target cell by the Pseudomonas exotoxin A translocationdomain II. The translocation leads the fusion antigen to entry into thetarget cell.

[0066] In Action (e), the carboxyl terminal moiety of the fusion antigenbinds to the ER membrane of the target cell for retention of the fusionantigen in the ER lumen for the process of the fusion antigen.

[0067] In Action (f), the antigenic moiety is processed in the ER lumen.The process includes, but is not limited to, antigen modification suchas glycosilation and proper digestion by enzyme in the ER lumen.

[0068] In Action (g), the processed fusion antigen can bind to a MHCclass I molecule. The MHC class I molecule itself is an uncompletedfolding protein and binds to many chaperones. The processed fusionantigen binds to the peptide-binding cleft to complete folding andstimulates the release of the chaperones.

[0069] In Action (h), the processed antigenic moiety is presented to thetarget cell surface by the MHC class I molecule. The folded MHC class Iand processed antigenic moiety is delivered to the cell surface.

[0070] In Action (i), the processed antigenic moiety carried by the MHCclass I molecule was recognized by CD8⁺T-cell to obtain an immunemessage for the recognition of the cytotoxic T-cell and also for thestorage an immune message into memory T-cells. Examples of the memoryT-cells are T_(cm) and T_(em) cells.

[0071] In Action (j), the immune message is stored by memory T-cells forimmunizing the animal. When the animal immunized with the fusion antigenis infected by the same antigen again, the memory T-cells evoke astronger immune response in a shorter time. T-cell vaccine provides anendogenous processing antigen which can be processed in the ER lumen ofthe target cell.

[0072] The present also provides a fusion porcine reproductive andrespiratory syndrome virus (PRRSV) ORF 7 antigen comprising a PRRSV ORF7 moiety; a Pseudomonas exotoxin A binding domain I; a Pseudomonasexotoxin A translocation domain II; and a carboxyl terminal moiety whichpermits retention of the fusion antigen in the endoplasmic reticulum(ER) membrane of a target cell.

[0073] A pharmaceutical composition comprising the fusion antigen of theinvention together with a pharmaceutically acceptable carrier is alsoprovided.

[0074] Another aspect of the invention is to provide a method ofimmunizing an animal for the prevention of porcine reproductive andrespiratory syndrome virus (PRRSV), which comprises the steps of:

[0075] (a) providing a fusion antigen comprising a PRRSV ORF 7 antigenmoiety, a Pseudomonas exotoxin A binding domain I, a Pseudomonasexotoxin A translocation domain II, and a carboxyl terminal moiety whichpermits retention of the antigen in the endoplasmic reticulum (ER)membrane of an target cell; and

[0076] (b) inoculating the fusion antigen into the animal.

[0077] The following examples are given for the purpose of illustrationonly and are not intended to limit the scope of the present invention.

EXAMPLE 1 PRRSV ORF 7 Fusion Antigen

[0078] Antigenic moiety: The nucleoprotein PRRSV ORF 7 gene sequence wasas shown in SEQ ID NO: 1 (Genbank Acc. No. AF035409, also shown in FIG.2). The PRRSV ORF 7 gene was cloned with specific primers, which werethe forward primer, 5′-GTC ACA TAT GCC AAA TAA CAA CGG CA-3′ (SEQ ID NO:2) and the reversed primer, 5′-AAG AAT TCC AGC TCA TCC ATG CTG-3′ (SEQID NO: 3). An Aat II restriction enzyme recognition site was used forligation and insertion to a Pseudomonas exotoxin A translocation domainII.

[0079]Pseudomonas exotoxin A binding domain I and Pseudomonas exotoxin Atranslocation domain II: pJH4 was used as a starting plasmid, whichencodes the Pseudomonas exotoxin A (PE) full-length gene as described byLiao C. W. et al. (Liao C. W. et al., 1995. Applied Microbiol Biotechnol43: 498-507). The Nde I-Eco R I DNA fragment of the full length of PEgene including domain I, II and III was constructed into pET15derivative plasmid to form a pET-PE plasmid, which has one Eco R I andXho I restriction enzymes recognition sites at the 3′-end of PE gene.

[0080] Fusion PE(ΔIII) with antigen gene: The 192 bp of Aat II-Eco R Ifragment (named D fragment), which is a C terminal DNA fragment of PRRSVORF 7 nucleoprotein gene, was obtained by RT-PCR and digested with AatII and Eco R I restriction enzymes. The DNA fragments were then purifiedby gel electrophoresis and electro-elution. This D fragment and the 7.1kb Aat II-Eco R I large fragment of pET-PE plasmid were ligated with T4ligase to form a plasmid pET15-H6-PE(ΔIII)-PRRS7-D (7.31 kb). Theplasmid comprised Eco R I and Xho I recognition sites at the end of thefusion gene. For increasing antigenicity, a DNA fragment with two tandemrepeated D fragments connected with a bridge (g) was created (named DGD)according to recombinant technique. A 7.7-kb plasmid encodingPE(ΔIII)-PRRS7-DgD (as shown in FIG. 3) was then constructed by theligation of the Sal I-DgD-Pst I fragment with a 6.0-kb Sal I-Pst Ifragment of pET15-H6-PE(ΔIII)-PRRS7-D (named PE-DGD).

[0081] Carboxyl terminal moiety: Gene sequence coding KKDELRDELKDEL (SEQID NO: 4) was shown in SEQ ID NO: 5. A Sal I restriction enzymerecognition site at 5′ end and a stop codon TAA-TGA at 3′ end were alsocreated (as shown in FIG. 4) and inserted into pET 23a plasmid digestedwith Nde I and Xho I to form pET23-3KDEL plasmid (as shown in FIG. 5).Synthesizing a polynucleotide encoding Sal I site-KKDELRDELKDEL-stopcodon-Xho I-Eco R I sequence through serial polymerase chain reaction(PCR). The linear DNA of pET23 cutting with Sal I as PCR DNA template, a168 bp fragment of first PCR product was generated by T7 promoter primerand a reversed primer 5′-TTC ATC TCT CAG TTC GTC TTT TTT GAG GTA GTC GACGGA GCT CGA ATT CGG-3′ (SEQ ID NO: 6). This DNA product contained a SalI recognition site. Then, the 168 bp of DNA as a second PCR template,one 206 bp fragment of a second PCR product was generated by the T7promotor primer and a reversed primer 5′-A GAA TTC CTC GAG TCA TTA CAGTTC GTC TTT CAG TTC ATC TCT CAG TTC GTC-3′ (SEQ ID NO: 7). The final PCRDNA fragment containing Sal I, Xho I, and Eco R I sites was obtained.The PCR-amplified DNA fragments were cleaved with Sal I and Eco R I andthen purified by gel electrophoresis and electro-elution. The purifiedSal I-Eco R I DNA fragments were ligated to the 3.6 kb of Sal I-Eco R IDNA fragment obtained from pET23a. Finally, the plasmid pET23-SalI-3KDEL, encoding SalI-KKDELRDELKDEL-stop codon-Xho I-Eco R I, wasconstructed.

[0082] Fusion antigen: The PRRSV ORF 7 fusion antigen was shown in FIG.1.

[0083] Two DNA fragments were prepared. One 6.4-kb Pst I-Xho I fragmentfrom plasmid pET15-H6-PE(ΔIII)-PRRS7-DgD (7.7 kb) containing PE(ΔIII)and antigen was obtained by digesting with Pst I and Xho I. Another1.345-kb Pst I-Xho I fragment containing the carboxyl terminal moietywas also obtained by digesting plasmid ET23-3KDEL with Pst I and Sal Irestriction enzymes. These two fragments were purified and then ligatedby T4 ligase to form pET23-H6-PE(ΔIII)-DgD-3KDEL (named PE-DGDk; asshown in FIG. 5).

[0084] Protein expression and purification: E. coli BL21(DE3)pLys cellsharboring plasmid for the expression of PE-DGD and PE-DGDk moleculeswere cultured in Luria Bertani broth containing 100 to 500 ppm ofampicillin at 37° C. When the culture attending early log phase,(A600=0.1 to 0.4), isopropyl-1-thio-β-D-galactopyranoside (IPTG) wasadded with a final concentration of 0.5 mM for induction. Cells wereharvested after induction after 2 hours and immediately stored at −70°C. The fusion antigen was partially purified by urea extraction asdescribed previously (Liao et al., 1995. Appl. Microbiol Biotechnol. 43:498-507). Under denaturing conditions, the PE-DGD and PE-DGDk moleculescontaining 6×His tag were fully exposed for improving binding to theNi-NTA matrix (Ni-NTA agarose; Qiagen® Lnc. Calif.). Therefore, theefficiency of the purification was maximized by reducing the potentialfor nonspecific binding. Batch purification of 6×His-tagged PE-DGD andPE-DGDk from E. coli under denaturing conditions was as described below:

[0085] adding 1 mL of the 50% Ni-TNA slurry to 4 mL lysate and mixinggently by shaking (e.g., 200 rpm) for 60 min at room temperature to forma lysate-resin mixture;

[0086] loading the lysate-resin mixture carefully into an empty columnwith the bottom cap still attached;

[0087] removing the bottom cap and collecting the flow-through solution;

[0088] washing twice with 4 mL wash buffer (100 mM NaH₂PO₄, 10 mMTris-HCl, 8 M urea, pH 8.0);

[0089] eluting the protein 4 times with 0.5 ml pH 5.9 elution buffer(100 mM NaH₂PO₄, 10 mM Tris-HCl, 8M urea, pH 5.9) followed by 4 timeswith 0.5 ml pH 4.5 elution buffer buffer (100 mM NaH₂PO₄, 10 mMTris-HCl, 8 M urea, pH 4.5); and

[0090] collecting fractions and subjecting to SDS-PAGE.

[0091] The results are shown in FIGS. 6 and 7. Quantitative analysis wasperformed using standard BSA protein. It showed that the fusion antigenwas successfully constructed.

EXAMPLE 2 PRRSV ORF 7 Fusion Antigen as T-Cell Vaccine

[0092] The preparation of PRRSV ORF 7 fusion antigen used herein aredescribed in Example 1.

[0093] Animals: Pigs were obtained from a herd periodically tested forPRRSV and known to be free of the virus by RT-PCR. Blood plasmafractions were collected. The RNA was extracted with a kit of NucleoSpinRNA II™ (Macherey-Nagel GmbH & Co. KG, Germany). RA1 solution of 350 μLand 3.5 μL of□β-mercaptoethanol were added into 100 μL plasma fractions.After reducing viscosity and clearing the lysate by filtration, thelysate was mixed with 350 μL of 70% ethanol. The RNA was adsorpted inNucleospin™ RNA column by centrifugation and followed by a wash.Ninety-five μL DNase solution was applied into the column for thedigestion of DNA. After repeating the wash and centrifugation severaltimes, RNA was eluted by 60 μL Rnase-free water.

[0094] RT-PCR was performed by using Qiagen Onestep RT-PCR Kit™ (Qiagen®Inc. Calif.). A forward primer of 5′-CCA GCC AGT CAA TCA GCT GTG-3′ (SEQID NO: 8) and a reverse primer of 5′-GCG GAT CAG GCG CAC-3′ (SEQ ID NO:9) were provided for synthesizing a 293-bp fragment. The detection limitof RT-PCR by agarose gel electrophoresis was determined around 10 ofPRRS (TCID₅₀/ml).

[0095] Blood leukocyte samples derived from five sows in a SPF farm weresubjected to RT-PCR. It showed that the five sows were not infected withPRRSV. The results are shown in FIGS. 8 to 10.

[0096] Immunization: Six new-born piglets were selected and individuallyidentified, weighed, and sexed from each of the three sows in a SPFfarm. The piglets were randomly sub-grouped into three groups ofvaccinated with PE-DGD, vaccinated with PE-DGDK and control based uponweight stratification, wherein each group comprised two piglets fromeach sow. In the vaccinated groups, intramuscular immunization wasperformed twice at the suckling stage. At the weaning stage(approximately 3 to 4 weeks of age), each group was removed and housedin an isolation room equipped with air conditioningand ventilation. Thevaccinated group was immunized twice at ages 4 and 18 days byintramuscular injection with 2 mL of vaccine containing 1 mL of PE-DGDor PE-DGDK (containing 50 μg protein/dose) emulsified in 1 mL ISA206(SEPPIC®, France), respectively. The control group was raised withoutimmunization.

[0097] Challenge in pig model: Two weeks after the final vaccination,the pigs were intranasally challenged after intramuscular administrationof 100 mg of Ketamine solution for sedation followed by intranasalinstillation of 1 ml of 2% Lidocaine for cough-reflex suppression. Fresh1 mL of MD-1 strain of PRRSV culture was used for challenge at doses ofabout 1×10⁷ TCID⁵⁰/mL. Five piglets were challenged in each group.

[0098] The blood leukocyte samples of the piglets were assayed withRT-PCR for detecting PRRSV two weeks after the second immunization andthe results are shown in FIG. 12. No viremia occurred in any of thepiglets before the PRRSV challenge. The blood leukocyte samples of thepiglets were again assayed with RT-PCR for detecting PRRSV after 3, 7,and 14 days after the challenge and the results are shown in FIGS. 13,14 and 15, respectively.

[0099] The results are also summarized in Table 1: TABLE 1 The PRRSVviremea ratio of piglets post-challenged with PRRSV Days Control PE-DGDkPE-DGD  3 3/5 3/5 3/5  7 3/4 (dead 1*) 2/5 2/4 (dead 1*) 14 3/3 (dead2*) 0/5 2/3 (dead 2*)

[0100] Necropsy was performed on all animals that had died and on allsurvivors at the end of the 2-week study. Macroscopic examinationrevealed pleuropneumonia in the lungs of five of the control pigs, fourof the pigs in PE-DGD vaccinated, and two of the pigs in PE-DGDK group.More extensive lesions were observed in the control and PE-DGDvaccinated but not in PE-DGDK vaccinated group. It showed that the PRRSVORF 7 fusion protein PE-DGDK can protect pigs from infection.

[0101] While embodiments of the present invention have been illustratedand described, various modifications and improvements can be made bypersons skilled in the art. It is intended that the present invention isnot limited to the particular forms as illustrated, and that all themodifications not departing from the spirit and scope of the presentinvention are within the scope as defined in the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 9 <210> SEQ ID NO 1 <211>LENGTH: 388 <212> TYPE: DNA <213> ORGANISM: Porcine reproductive andrespiratory syndrome virus <300> PUBLICATION INFORMATION: <308> DATABASEACCESSION NUMBER: Genbank/AF035409 <309> DATABASE ENTRY DATE: 1998-12-29<313> RELEVANT RESIDUES: (2898)..(3269) <400> SEQUENCE: 1 catatgccaaataacaacgg caagcagcag aagaaaaaga agggggacgg ccagccagtc 60 aatcagctgtgccaaatgct gggtaagatc atcgcccagc aaagtcagtc cagagttaag 120 ggaccgggaaggaaaaataa gaagaaaaac ccggagaagc cccattttcc tctggcgact 180 gaagatgacgtcagacacca ctttaccccc agtgagcggc aattgtgttt gtcgtcaatc 240 cagactgcctttaatcaagg cgctggaact tgcatcctgt cagattctgg gaggataagt 300 tacactgtggagtttagttt gcctacgcat catactgtgc gcctgatccg cgttacagca 360 ccaccctcagcataatgggc tggaattc 388 <210> SEQ ID NO 2 <211> LENGTH: 26 <212> TYPE:DNA <213> ORGANISM: ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: aforward primer for synthesizing PRRSV ORF 7 <400> SEQUENCE: 2 gtcacatatgccaaataaca acggca 26 <210> SEQ ID NO 3 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: aforward primer for synthesizing PRRSV ORF 7 <400> SEQUENCE: 3 aagaattccagctcatccat gctg 24 <210> SEQ ID NO 4 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: acarboxyl terminal moiety sequence in Example 1 <400> SEQUENCE: 4 Lys LysAsp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu 1 5 10 <210> SEQ ID NO 5<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL <220>FEATURE: <223> OTHER INFORMATION: a gene encoding the carboxyl terminalmoiety in Example 1 <400> SEQUENCE: 5 aaaaaagacg aactgagaga tgaactgaaagacgaactg 39 <210> SEQ ID NO 6 <211> LENGTH: 51 <212> TYPE: DNA <213>ORGANISM: ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: a reversedprimer for generating the first polymerase chain reaction in Example 1<400> SEQUENCE: 6 ttcatctctc agttcgtctt ttttgaggta gtcgacggag ctcgaattcgg 51 <210> SEQ ID NO 7 <211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM:ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: a reversed primer forgenerating the second polymerase chain reaction in Example 1 <400>SEQUENCE: 7 agaattcctc gagtcattac agttcgtctt tcagttcatc tctcagttcg tc 52<210> SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: a forward primer forRT-PCR detection of PRRSV <400> SEQUENCE: 8 ccagccagtc aatcagctgt g 21<210> SEQ ID NO 9 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:ARTIFICIAL <220> FEATURE: <223> OTHER INFORMATION: a reversed primer forRT-PCR detection PRRSV <400> SEQUENCE: 9 gcggatcagg cgcac 15

What is claimed is:
 1. A fusion antigen specific for a target cellcomprising: an antigenic moiety; a ligand moiety which is capable ofreacting, recognizing or binding to a receptor on the target cell; aPseudomonas exotoxin A translocation domain II; and a carboxyl terminalmoiety which permits retention of the fusion antigen in the endoplasmicreticulum (ER) membrane of the target cell.
 2. The fusion antigenaccording to claim 1, wherein the target cell is an antigen presentingcell.
 3. The fusion antigen according to claim 1, wherein the targetcell is selected from the group consisting of T-cells, B-cells,dendritic cells, monocytes, and macrophages.
 4. The fusion antigenaccording to claim 1, wherein the antigenic moiety is derived fromporcine reproductive and respiratory syndrome virus, Circovirus type II,or human immunodeficiency virus.
 5. The fusion antigen according toclaim 1, wherein the antigenic moiety is selected from the groupconsisting of porcine reproductive and respiratory syndrome virus(PRRSV) ORF 1, 2, 3, 4, 5, 6, and
 7. 6. The fusion antigen according toclaim 1, wherein the antigenic moiety comprises at least one antigenicunit and the adjacent antigenic unit is connected by a bridge region. 7.The fusion antigen according to claim 1, wherein the receptor to bebound to the ligand moiety is selected from the group consisting ofantibody receptors, growth factor receptors, lymphokine receptors,cytokine receptors, and hormone receptors.
 8. The fusion antigenaccording to claim 1, wherein the receptor to be bound to the ligandmoiety is selected from the group consisting of TGFα receptors, IL2receptors, IL4 receptors, IL6 receptors, IGF 1 receptors, CD4 receptors,IL18 receptors, IL 12 receptors, EGF receptors, LDL receptors andα2-macroglobulin receptors.
 9. The fusion antigen according to claim 1,wherein the ligand moiety is a Pseudomonas exotoxin A binding domain I.10. The fusion antigen according to claim 1, wherein the carboxylterminal moiety comprises, in a direction from the amino terminus to thecarboxyl terminus, the following amino acid residues:R¹-R²-R³-R⁴-(R⁵)_(n) Wherein, R¹ is a positively charged amino acidresidue; R² is a negatively charged amino acid residue; R³ is anegatively charged amino acid residue; R⁴ is L; R⁵ is a positivelycharged amino acid residue; and n is 0 or
 1. 11. The fusion antigenaccording to claim 1, wherein the carboxyl terminal moiety comprises asequence of KDEL.
 12. The fusion antigen according to claim 1, whereinthe carboxyl terminal moiety comprises a sequence of KKDL-RDEL-KDEL. 13.A pharmaceutical composition comprising the fusion antigen according toclaim 1 together with a pharmaceutically acceptable carrier.
 14. Thepharmaceutical composition according to claim 13 is a T-cell vaccine.15. A method of immunizing an animal comprising the steps of: (a)providing a fusion antigen specific for a target cell comprising anantigenic moiety, a ligand moiety which is capable of reacting,recognizing or binding to a receptor on the target cell, a Pseudomonasexotoxin A translocation domain II, and a carboxyl terminal moiety whichpermits retention of the fusion antigen in the endoplasmic reticulum(ER) membrane of the target cell; and (b) inoculating the fusion antigeninto the animal.
 16. The method according to claim 15, wherein thetarget cell is an antigen presenting cell.
 17. The method according toclaim 15, wherein the antigenic moiety is derived from porcinereproductive and respiratory syndrome virus, Circovirus type II, orhuman immunodeficiency virus.
 18. The method according to claim 15,wherein the antigenic moiety is selected from the group consisting ofporcine reproductive and respiratory syndrome virus (PRRSV) ORF 1, 2, 3,4, 5, 6, and
 7. 19. The method according to claim 15, wherein theantigenic moiety comprises at least one antigenic unit and the adjacentantigenic unit is connected by a bridge region.
 20. The method accordingto claim 15, wherein the ligand moiety is a Pseudomonas exotoxin Abinding domain I.
 21. The method according to claim 15, wherein thereceptor to be bound to the ligand moiety is selected from the groupconsisting of antibody receptors, growth factor receptors, lymphokinereceptors, cytokine receptors, and hormone receptors.
 22. The methodaccording to claim 15, wherein the receptor to be bound to the ligandmoiety is selected from the group consisting of TGFα receptors, IL2receptors, IL4 receptors, IL6 receptors, IGF 1 receptors, CD4 receptors,IL18 receptors, IL 12 receptors, EGF receptors, LDL receptors andα2-macroglobulin receptors.
 23. The method according to claim 15,wherein the target cell is selected from the group consisting of T cell,B cell, dendritic cell, monocyte, and macrophage.
 24. The methodaccording to claim 15, wherein the carboxyl terminal moiety comprises,in a direction from the amino terminus to the carboxyl terminus, thefollowing amino acid residues: R¹-R²-R³-R⁴-(R⁵)_(n) Wherein, R¹ is apositively charged amino acid residue; R² is a negatively charged aminoacid residue; R³ is a negatively charged amino acid residue; R⁴ is L; R⁵is a positively charged amino acid residue; and n is 0 or
 1. 25. Themethod according to claim 15, wherein the carboxyl terminal moietycomprises a sequence of KDEL.
 26. The method according to claim 15,wherein the carboxyl terminal moiety comprises a sequence ofKKDL-RDEL-KDEL.
 27. A fusion porcine reproductive and respiratorysyndrome virus (PRRSV) ORF 7 antigen comprising a PRRSV ORF 7 moiety; aPseudomonas exotoxin A binding domain I; a Pseudomonas exotoxin Atranslocation domain II; and a carboxyl terminal moiety which permitsretention of the fusion antigen in the endoplasmic reticulum (ER)membrane of a target cell.
 28. The fusion antigen according to claim 27,wherein the target cell is an antigen presenting cell.
 29. The fusionantigen according to claim 27, wherein the target cell is selected fromthe group consisting of T cell, B cell, dendritic cell, monocyte, andmacrophage.
 30. The fusion antigen according to claim 27, wherein theantigenic moiety comprises at least one antigenic unit and the adjacentantigenic unit is connected by a bridge region.
 31. The fusion antigenaccording to claim 27, wherein the carboxyl terminal moiety comprises,in a direction from the amino terminus to the carboxyl terminus, thefollowing amino acid residues: R¹-R²-R³-R⁴-(R⁵)_(n) Wherein, R¹ is apositively charged amino acid residue; R² is a negatively charged aminoacid residue; R³ is a negatively charged amino acid residue; R⁴ is L; R⁵is a positively charged amino acid residue; and n is 0 or
 1. 32. Thefusion antigen according to claim 27, wherein the carboxyl terminalmoiety comprises a sequence of KDEL.
 33. The fusion antigen according toclaim 27, wherein the carboxyl terminal moiety comprises a sequence ofKKDL-RDEL-KDEL.
 34. A pharmaceutical composition comprising the fusionantigen according to claim 27 together with a pharmaceuticallyacceptable carrier.
 35. The pharmaceutical composition according toclaim 34 is a T-cell vaccine.
 36. A method of immunizing an animal forthe preventing porcine reproductive and respiratory syndrome virus(PRRSV), which comprises the steps of: (a) providing a fusion antigencomprising a PRRSV ORF 7 antigen moiety, a Pseudomonas exotoxin Abinding domain I, a Pseudomonas exotoxin A translocation domain II, anda carboxyl terminal moiety which permits retention of the antigen in theendoplasmic reticulum (ER) membrane of a target cell; and (b)inoculating the fusion antigen into the animal.
 37. The method accordingto claim 36, wherein the target cell is an antigen presenting cell. 38.The method according to claim 36, wherein the target cell is selectedfrom the group consisting of T-cells, B-cells, dendritic cells,monocytes, and macrophages.
 39. The method according to claim 36,wherein the antigenic moiety comprises at least one antigenic unit andthe adjacent antigenic unit is connected by a bridge region.
 40. Themethod according to claim 36, wherein the carboxyl terminal moietycomprises, in a direction from the amino terminus to the carboxylterminus, the following amino acid residues: R¹-R²-R³-R⁴-(R⁵)_(n)Wherein, R¹ is a positively charged amino acid residue; R² is anegatively charged amino acid residue; R³ is a negatively charged aminoacid residue; R⁴ is L; R⁵ is a positively charged amino acid residue;and n is 0 or
 1. 41. The method according to claim 36, wherein thecarboxyl terminal moiety comprises a sequence of KDEL.
 42. The methodaccording to claim 36, wherein the carboxyl terminal moiety comprises asequence of KKDL-RDEL-KDEL.